The expression of recombinant genes in bacterial hosts is most often achieved by the introduction of episomal self-replicating elements (e.g. plasmids) that encode the structural gene of the protein of interest under the control of an appropriate promoter, into host bacteria. Such plasmids are most commonly maintained by the inclusion of selective marker genes that encode proteins that confer resistance to specific antibiotics (such as ampicillin, chloramphenicol, kanamycin, tetracycline etc.). They are then maintained in the host by addition of the appropriate antibiotic to the culture medium.
Stable maintenance of plasmids in host strains often requires the addition of the appropriate antibiotic selection without which they may segregate out giving rise to significant numbers of cells in any culture, that are devoid of plasmid and therefore cannot express the desired product.
However, the use of antibiotics in the production of recombinant proteins is undesirable for a number of reasons. Apart from the obvious increase in costs arising from the need to add them as a supplement to the growth medium, the use of antibiotics is considered a problem in the production of any recombinant protein intended for human or veterinary use. This is primarily for three reasons. Firstly, residual antibiotics can, in sensitive individuals, cause severe allergic reactions. Secondly, there is the possibility of selection for antibiotic resistant bacteria in the natural bacterial flora of those using the product, and finally, DNA encoding the antibiotic resistance may also be transferred to sensitive bacteria in individuals using the product, thereby also spreading undesired antibiotic resistance in a cohort.
There are already inventions dealing with this problem, one such is the par gene which will effectively kill all cells that do not retain a copy of the plasmid after each cell division [1].
Another patent application [2], which touches on the invention described herein, was based on the knowledge of the thyA DNA sequence in E. coli. The authors introduced the thyA gene on a plasmid but used host strains that were spontaneous thyA− mutants selected on the bases of trimethoprim resistance. Such mutants are not well defined (carrying point mutations or small deletions) and may revert to the wild-type (i.e. thyA+) at unacceptably high frequencies. This would lead to that the host bacteria could eliminate the plasmid and hence lose, or not give consistent and reliable, production of the desired recombinant product. An additional problem with trimethoprim selection is the possibility that resulting thymine dependence may arise due to a mutation in the dihydrofolate reductase (folA) gene and hence not be complemented by a plasmid-borne thyA gene [3]. This patent application has been discontinued at least in Europe.
The use of V. cholerae for expression of recombinant genes has been shown to be advantageous over other prokaryotic expression systems in common use in that specific recombinant products may be produced in large quantities and secreted into the culture medium, thereby facilitating downstream purification procedures. This is in contrast to E. coli where the product often assembles in the periplasmic space [4]. One important factor endowing V. cholerae with this property is the eps genes in V. cholerae [5].
Thymidylate synthetase encoded by the thyA gene of Escherichia coli and other bacteria catalyses the methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) and is an essential enzyme in the biosynthesis of deoxyribothymidine triphosphate (dTTP) for incorporation into DNA. In the absence of this enzyme the bacteria become dependent upon an external source of thymine which is incorporated into dTTP by a salvage pathway encoded by the deo genes [6].
Spontaneous mutants that are thyA− can be readily isolated on the basis of trimethoprim resistance. This antibiotic inhibits tetrahydrofolate regeneration from dihydrofolate produced by thymidylate synthetase-catalysed dTMP synthesis. Thus, if the cells are thyA they become thymine dependent but no longer deplete the tetrahydrofolate pool in the presence of trimethoprim.